Assessment for acceleration and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma using histologic and immunohistochemical features: a case series

Morphologic features of aggressive/ “accelerated” chronic lymphocytic leukemia/small lymphocytic lymphoma (aCLL/SLL) have been described. Richter transformation (RT) also occurs in a subset of CLL/SLL cases. This case series examined inter-observer variability when assessing for aCLL/SLL and RT, with attention to how immunohistochemical (IHC) markers may assist in this evaluation. Twelve cases of CLL/SLL with available FFPE tissue were identified. H&E staining and IHC (CD3, CD20, CD5, CD23, LEF1, LAG3, C-MYC, PD-1, MUM1, Cyclin D1, BCL-6, p53, and Ki-67) were performed. Three hematopathologists reviewed each case. The pathologists provided a final interpretation of (1) CLL/SLL, (2) CLL/SLL with expanded and/or confluent proliferation centers or increased Ki-67 (aCLL/SLL), or (3) large cell transformation/DLBCL. The pathologists lacked consensus in the diagnosis in 6/12 cases (50%). The reviewers disagreed on the presence of expanded/confluent proliferation centers in 8/12 cases (67%). With the exception of Ki-67, no IHC marker showed a difference in the staining profile in aCLL/SLL or RT compared to low-grade cases. This series showed inter-observer variability in the evaluation for aCLL/SLL and RT. A study that serially examines genetic alterations in FFPE tissue and correlates the features with histology and IHC, at diagnosis and throughout the disease course, may help refine indicators of aggressive disease.


Introduction
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) commonly demonstrates "classic" or "typical" histologic features in tissue specimens.A monotonous population of small lymphocytes with scant cytoplasm, round nuclear contours, and condensed chromatin effaces lymph node architecture, with scattered small proliferation centers [1,2].Richter transformation (RT) of CLL/SLL is characterized by diffuse large B-cell (DLBCL) morphology or, less commonly, classic Hodgkin lymphoma (CHL) morphology [3,4].CLL/SLL with "accelerated" or "aggressive" features (aCLL/SLL) is utilized to describe cases with morphology and clinical behavior in-between classic CLL/ SLL and RT.Histologically, this is commonly defined using the criteria suggested by Giné et al. in 2010, including the presence of proliferation centers broader than a 20× field and/or the presence of a high proliferation rate, as indicated by Ki-67 greater than 40% or mitotic rate greater than 2.4 within proliferation centers [5].However, there are other described patterns of aCLL/SLL, to include cases with increased intermediate-to large cells (prolymphocytic or paraimmunoblastic morphology) growing in a scattered or dispersed pattern, without clearly identifiable pseudofollicles or proliferation centers [2,6].
This study sought to gauge the degree of pathologist interobserver variability when assessing for features of aCLL/ SLL and RT.We integrated multiple previously reported immunohistochemical (IHC) markers to determine how they may aid in this assessment.Cytogenetic and next-generation sequencing (NGS) data were available for select cases, demonstrating the challenges of integrating the histologic features with ancillary genetic studies.

Case selection
Institutional surgical pathology archives were queried from 2016 to 2022.Cases selected for inclusion were required to have available formalin-fixed-paraffin-embedded (FFPE) tissue and evidence of CLL/SLL by compelling clinical, histologic, and/or flow cytometry evidence, as determined by a pathologist (MM) upon review of pathology reports and the electronic medical record.Select cases with what was previously described as low-grade or "classic" morphology were chosen (cases 1-5), with available whole-tissue sections (as opposed to core biopsies).All cases with previously documented atypical/accelerated/"high-grade" features or known Richter transformation were included.In cases with a predominance of large cells, diffuse large B-cell-like morphology, or "Richter transformation" noted in the pathology report, patients were required to have a well-documented history of CLL/SLL (by prior documented flow cytometry or histology) to be included.Cases of transformation with non-DLBCL morphology (Hodgkin lymphoma, plasmablastic lymphoma, etc.) were not included.
Clinical information was extracted from the medical record, as available.Rai/Binet/Ann Arbor staging was not consistently documented at the time of biopsy and therefore was not included for consideration.Results (prior and concurrent) of TP53 mutation testing, IgHV hypermutation testing, karyotype analysis, and fluorescence in situ hybridization (FISH) performed on peripheral blood were noted when available.
Evaluations were performed on BX51 and BX53 Olympus microscopes (all with 20 × objective of CN20x/0.50 and an ocular WHN 10x/22, Evident/Olympus Scientific Solutions).Reviewers were required to score cases using a standardized worksheet with a checklist of H&E and IHC features.Reviewers documented the quality of the H&E preparation, the presence/absence of identifiable proliferation centers, and the presence/absence of expanded (broader than 20 × field) or confluent proliferation centers, according to the criteria outlined by Giné et al. [5].An average mitotic rate (by H&E examination) across 10 proliferation centers was calculated, if proliferation centers were deemed identifiable.If proliferation centers were not readily identifiable, an average mitotic index was calculated across 10, 40x (0.238 mm 2 ) high-power fields.DLBCL/RT was defined as diffuse sheets or nodules of large cells, in keeping with DLBCL morphology outlined in the World Health Organization Classification of Haematolymphoid Tumours, 5th edition [1].

OncoScan and next-generation sequencing
DNA extraction was performed on FFPE tissue from select cases using a QIAamp DNA FFPE tissue kit, per manufacturer specifications.OncoScan microarray (OncoScan, Ther-moFisher) and PGDx elio™ tissue complete next-generation sequencing (NGS, 505 gene panel) analyses were performed on FFPE samples from select cases using manufacturer's recommended protocols.Analysis of OncoScan and PGDx data was performed by molecular pathology faculty according to professional guidelines [19,20].

Results
Twelve cases were identified: 11 excisions and one large core biopsy (Table 1).Specimen types included lymph node (n = 10), tonsil (n = 1), and submandibular gland (n = 1).Two biopsies were serial specimens from the same patient (specimens 7 and 8).The three reviewing pathologists showed consensus in the final diagnosis for 6 of 12 (50%) cases and agreed with the historic interpretation in 5 cases (42%, Fig. 1, Panel 3).While no two pathologists consistently agreed or disagreed on all cases, reviewer three did demonstrate a trend of lower scoring compared to the other two reviewers.A reviewer was the historic pathologist on record in 7 cases (58%).Despite a washout period and blinded case numbers, when these pathologists re-reviewed cases, there were no changes in diagnosis between the historic diagnosis and current evaluation in all 7 cases.
The three reviewers disagreed on the presence or absence of clearly delineated proliferation centers in 8 of 12 cases (67%).Furthermore, there was lack of consensus in the presence or absence of expanded (> 20x) proliferation centers in 8 of 12 cases (67%).Reviewers commented on challenges associated with the H&E evaluation of select cases, including fixation artifact impeding accurate evaluation, difficulty in identifying proliferation centers in non-nodal tissue, and variant patterns in which dispersed large cells were identified but true proliferation centers were challenging to appreciate (Fig. 2).Mitotic rate assessment was also variable across reviewers, including 4 cases (33%) in which one reviewer fell on a different side of the suggested 2.4 mitoses/ proliferation center (or high-power field) cutoff compared to other reviewers (Fig. 1, Panel 2).Ki-67 assessment was discrepant in 4 cases (33%), in which one reviewer fell on a different side of the 40% cutoff for aCLL/SLL.
Interpretation of immunohistochemical stains for CD20, CD5, and CD23 was largely consistent across reviewers.Cyclin D1 was interpreted as negative in the B cell population by all three reviewers in the majority of cases.Two cases (1 and 10) had Cyclin D1 staining in proliferation centers, though this was not consistently reported across reviewers.The interpretation of the remaining immunohistochemical markers (LEF1, LAG3, C-MYC, PD1, MUM1, BCL-6, and p53) was variable between observers (Fig. 1, Panel 1).Furthermore, the observation of increased expression in proliferation centers was inconsistent across reviewers.While there were insufficient cases to enable formal statistical analysis, with the exception of Ki-67 showing increased staining in cases with more aggressive histology, no marker showed a clear difference in the staining profile in aCLL/SLL or RT compared to low-grade cases.This remained true when assessing differential expression within proliferation centers.
Cases 7 and 8 (serial specimens from the same patient over a period of approximately three months) showed < 1% (null pattern) staining for p53, which showed concordant scoring across all three reviewers; del(17p) was detected by peripheral blood FISH/karyotype and OncoScan performed on FFPE tissue.In addition, case 12 was scored as having > 80% staining for p53 by all three reviewers, though confirmatory p53 testing was not performed in the clinical workup for this patient.For the remaining 9 cases, p53 staining pattern did not show a clear correlation with available molecular and cytogenetic data, though dedicated TP53 mutational testing was only performed in a minority of cases.
Historic FISH/karyotype testing (performed prior to collection of the specimen under review) was reported for 4 (33%) cases (Table 1).OncoScan was performed on the current, FFPE specimen in 6 cases (50%, cases 3, 4, 5, 7, 9, and 10).NGS was performed on the current, FFPE specimen in 2 cases (9 and 10) demonstrating a NOTCH1 frameshift mutation and PTPN11 p.S502T mutation in case 9 and KRAS G12V mutation in case 10.Concurrent FISH/Karyotype (performed at the same time as the tissue biopsy) was performed on peripheral blood in 4 cases (3, 5, 8, and 10).Concurrent TP53 mutation testing and IgHV hypermutation testing were performed on peripheral blood in 2 cases (5 and 10).

Discussion
This study demonstrates considerable inter-observer variability in the evaluation for aCLL/SLL and RT, even when employing a systematic approach with suggested criteria.This variability was observed in examination of H&E features, evaluation for indices of proliferation rate (mitotic index and Ki-67), and the evaluation of additional immunohistochemical stains previously suggested to have prognostic value in the literature.While Ki-67 showed an expected trend of increased staining in cases with more aggressive morphology, none of the additional immunohistochemical stains showed clear utility in this evaluation.This is a small case series, and there may be a degree of ascertainment bias due to the case selection criteria.Only a subset of low-grade/classic cases with available whole-tissue sections are represented, and the search methods utilized to identify cases with aggressive morphology or transformation have a risk of enriching for cases with challenging morphology.Another potential limitation of the study was the inability to assess for immunohistochemical staining of p27, a cyclin-dependent kinase inhibitor that is negative in proliferation centers, which was stated to have utility by Giné et al. in delineating and measuring proliferation centers [5].This immunohistochemical stain is not available at our institution.However, given the variability across pathologists in almost all aspects of histologic review, we believe it unlikely that incorporating analysis of p27 would resolve all of the observed discrepancies in interpretation.
This series nevertheless highlights the challenge of assigning discrete diagnoses to what likely represents a continuous spectrum of disease [3,21].This challenge is compounded by other variables, such as the amount of tissue present for evaluation and the need to evaluate both nodal and non-nodal sites of disease.Artifacts of tissue processing and staining can also contribute to diagnostic uncertainty, and laboratories would be wise to mitigate this influence by optimizing protocols for lymph node specimens to ensure high quality morphologic evaluation.Accompanying this area of diagnostic uncertainty, the clinical implications of assigning these histologic categories is not always clear.Management implications of rendering a diagnosis of aCLL/SLL versus classic CLL/SLL are not well defined.Current National Comprehensive Cancer Network (NCCN) guidelines provide no definitive provisions on management of aCLL/SLL, with patients being managed with standard CLL/SLL regimens, on clinical trials, or according to provider discretion [22].Histologic features of RT may trigger transition to more aggressive therapy [22].However, sampling of proliferation centers in limited core biopsies has previously been shown to be a risk of over-interpretation as evidence of RT [21].Furthermore, recognition of "pseudo-transformation" in the setting of modern Bruton's tyrosine kinase inhibitor therapy further indicates that histology as an isolated finding can be an imperfect predictor of disease behavior [23].Our study demonstrates that distinction between aCLL/SLL and RT can be difficult.Cases 7 and 8, samples taken from a single patient over an approximately three-month period, illustrate how differences in interpretation may impact clinical management.The first biopsy (case 7) was originally diagnosed as aCLL/SLL by the historic reviewer and venetoclax was initiated, though two other pathologists, on retrospective review for this study, considered the features compatible with RT.Likewise, a follow-up biopsy (case 8) was considered diagnostic for RT by the historic pathologist, a diagnosis factored into a decision to initiate R-CHOP therapy, but other reviewing pathologists retrospectively characterized the case as aCLL/SLL.Given these challenges, utilization of a "consensus" review system at the time of diagnostic evaluation may be a helpful strategy, with multiple hematopathologists reviewing the case and arriving at a group/consensus diagnosis in instances when histology is not straightforward or a change in diagnosis is expected to impact therapy.While genetic alterations of CLL/SLL associated with adverse clinical behavior have been repeatedly studied, correlation of these genetic signatures with aggressive histologic features remains imperfect [24,25].This is compounded by molecular and cytogenetic studies of CLL/ SLL, in both clinical and research settings, being commonly performed on peripheral blood or bone marrow specimens, as opposed to FFPE tissue [26].The ability to definitively correlate genetic signatures with aggressive histology is beyond the scope of this study, as there are too few cases and NGS and cytogenetic data are only available for a subset of specimens.However, this series prompts re-examination of the relative weight that histology and immunohistochemistry should bear in an evergrowing menu of prognostic data elements (flow cytometry immunophenotype, IgHV hypermutation status, and cytogenetic and molecular abnormalities).As correlation of genetic and histologic features remains limited, a larger study that serially examines genetic alterations in FFPE tissue in conjunction with histology, from initial diagnosis and throughout the disease course, may be helpful in establishing indicators of evolving or progressive disease to help refine clinical action points.Intermediate-power examination demonstrates confluence of the palestaining areas, while high-power examination demonstrates abundant large cells in small sheets, though some small lymphocytes are still appreciable.Ki-67 (inset) was variable, with some foci showing up to 60% staining

Fig. 1 Fig. 2
Fig. 1 Comparison of immunohistochemistry results, proliferation indices, and final diagnosis, by reviewer.Immunohistochemical (IHC) stains and final diagnosis, as scored by 3 reviewers.Ki-67 score reflects entire range identified in case (including outside and within proliferation centers, when proliferation centers identified).Mitotic

Table 1
Patient characteristics and results of ancillary studies.Ancillary studies are summarized according to test performed and specimen type used for analysis, designated in parentheses